The present invention relates to the field of the production of transgenic plants through Agrobacterium-mediated transformation of cells of somatic embryogenic calli or embryogenic suspension cultures and regeneration of the transformed cells into fruit-setting plants. In particular, the present invention relates to the production of transgenic plants in the Euphorbiaceae family. The present invention further relates to media compositions, selection methods and engineered Agrobacterium tumefaciens strains that improve Agrobacterium-mediated transformation efficiency.
The publications and other materials used herein to illuminate the background of the invention or provide additional details respecting the practice, are incorporated by reference, and for convenience are respectively grouped in the Bibliography.
The world is facing dwindling supply is fossil fuel and worsening Green House Effect. There is an urgent demand to increase production and consumption of renewable energy. Biofuels have been recognized as a national priority for many countries in their search for alternative sources to meet their energy security needs and at the same time help reduce CO2 emissions that cause the Green House Effect. The demand for biofuel has put increasing pressure on food production. For example, to satisfy the biofuel need for Germany in 2017 as mandated by the German government the entire farm land of this country would have to be used for growing bioenergy crops with no land left for food production. To ease this competition for land and to satisfy our need for renewable fuels, there is a strong need to utilize marginal land for bio-energy production.
Euphorbiaceae family includes valuable trees species such as rubber (Hevea brasiliensis) and Jatropha curcas and castor oil plant (Ricinus communis). Several unique characteristics of Jatropha curcas make it an ideal plant for biodiesel production. These characteristics include the ability to grow on marginal land; low requirement for water; a non-food crop status; fast oil production in 0.5-2 years after planting compared to more than 3.5 years for oil palm. Accordingly, the Indonesian government has announced that they will dedicate about 3 million hectares of land for jatropha planting in the next 5 years. Many other Asian countries are adopting similarly bold plans. Amongst the various countries, India is the most advanced in terms of establishment of Jatropha plantations. However, the seed yield of an Indian Jatropha plantation remains low, ranging from 0.4 to 12 MT/Ha (compared with about 19Mt/Ha for palm fruit). Similarly, the reported kernel oil contents varied wildly from 40.4-85.3% (Sun, W. B., Jatropha International Congress 2008, Singapore). Castor oil is an important source of lubricant and is also a potential source of biodiesel in the future.
This difference is at least in part attributed to the lack of research in breeding and farm management in Jatropha curcas. Genetic engineering is recognized as a fast method for crop improvement. Plant transformation is essentially a two step process, i.e., delivery of genes into a host cell followed by regeneration of the transformed cell into a plant. Somatic embryogenic calli or somatic embryogenic suspension cultures is generally regarded as the most efficient method of regeneration as most of the transformed cells have already acquired the embryogenic potential that will drive them to develop into a somatic embryo quite spontaneously. An efficient method for initiation of Jatropha somatic embryos and maintenance of embryogenic suspension cultures have been described (International patent application No. PCT/SG2009/000015; US Provisional Patent: 61/025,430).
Plant transformation methods may be broadly classified into two groups: Direct DNA transfer, such as electroporation and particle bombardment, and Agrobacterium-mediated transformation. To date, the later has become method of choice in transformation of many plants. In fact, most commercially available transgenic varieties of cotton and rapeseed derived from this method (Cerdeira and Duke, 2006; Dunwell, 2000). Previous work in maize (Dunwell, 1999; Vega et al., 2008; Wang and Frame, 2009), rice (Christou, 1997; Hayashimoto et al., 1990; Lee et al., 1991; Zhang et al., 1997) and soybean (Christou et al., 1988; Christou et al., 1987; Olhoft et al., 2003) demonstrate different examples of different methods for introduction of foreign DNA into crop plants. Transgenic plants may be obtained following Agrobacterium-mediated transformation of the. This method is characterized by a low frequency of transgenic production and by the formation of chimeras (Gould et al., 1991). Transgenic plants may also be obtained following Agrobacterium-mediated transformation of immature embryos. Immature embryos have been the choicest explant to date since there is usually a very high frequency of callus induction and plant regeneration from immature embryos. Following transformation, immature embryos, regardless of the method of DNA delivery, are very hard to regenerate into fertile plants (Cheng et al., 1997). In addition, it is usually very difficult to obtain immature embryos throughout the year, and their suitable stage for culture is also strictly time dependent, thus limiting their use. While the frequency of transformation with immature embryos ranges from about 0.14 to about 4.3%, the number of transgenics recovered is a small fraction of the number of embryos transformed (Cheng et al., 1997). Somatic embryos are suitable for transformation via Agrobacterium tumefaciens and particle bombardment (Christou, 1997; Sidorov and Duncan, 2009). Somatic embryogenic calli can be maintained conveniently as a liquid culture and fast-amplified for production of clonal populations. Thus, once a somatic embryogenic culture of an elite variety of established, it will save years of time developing genetically pure lines that are stacked with transgenic traits.
Many different explants can be co-cultured with an Agrobacterium strain harboring a T-DNA vector to produce a transgenic cell, which can be regenerated into a normal plant through somatic embryogenesis or organogenesis pathway. Research on regeneration and transformation of Jatropha curcas has been very limited.
Jha et al. (2007) reported production of somatic embryos of Jatropha curcas using leaf tissues. (Li et al., 2008) report transformation of Jatropha curcas by co-culturing leaf disc with Agrobacterium and regenerate via organogenesis pathway. However, both methods are not easily repeatable in the Jatropha research community (Z. Mao et al., Temasek Life Sciences Laboratories, Singapore, peronsonal communication; A. Suwanto, Bogor Agricultural University, Indonesia; personal communication).
Mao et al. (U.S. provisional patent application No. 61/122,454) discloses production of transgenic Jatropha curcas plants by Agrobacterium-mediated transformation of leaf explants and regeneration via somatic embryogenesis pathway. The invention also used grafting technique to overcome rooting difficulties of the transgenic plantlets.
International Publication No. WO 2008/012832 discloses an efficient process for in vitro propagation of Jatropha through direct regeneration of leaf disc without any intermediary callus phase. Genetic transformation based on this regeneration method has not been reported.
Thus, a reliable and efficient method for transformation of Jatropha is yet to be developed. Improvements that would lead to shorter regeneration time, higher transformation rate and high throughput transformation work are particularly welcome.
Delivery of T-DNA by Agrobacterium cells into plant host cells depends on pre-induction of virulence genes. Known inducers that are naturally produced in wounded plant tissues include a variety of phenolic compounds such as acetosyringone, sinapinic acid (3,5 dimethoxy-4-hydroxycinnamic acid), syringic acid (4-hydroxy-3,5 dimethoxybenzoic acid), ferulic acid (4-hydroxy-3-methoxycinnamic acid), catechol (1,2-dihydroxybenzene), p-hydroxybenzoic acid (4-hydroxybenzoic acid), β-resorcylic acid (2,4 dihydroxybenzoic acid), protocatechuic acid (3,4-dihydroxybenzoic acid), pyrrogallic acid (2,3,4-trihydroxybenzoic acid), gallic acid (3,4,5-trihydroxybenzoic acid) and vanillin (3-methoxy-4-hydroxybenzaldehyde) (U.S. Pat. No. 6,483,013).
A constitutively expressed virG mutant protein (virGN54D) has been found to enhance Agrobacterium virulence and transformation efficiency (Hansen et al., 1994). Similarly, supplementation of virulence inducers, e.g., acetosyringone or nopaline, increased transformation efficiency of cotton shoot tips (Veluthambi et al., 1989) and somatic embryogenic callus that was propagated on solid media (U.S. Pat. No. 6,483,013). Acetosyringone is indispensable for transformation of a large number of fungi, which are unable to secrete Agrobacterium virulence inducing compounds (Bundock et al., 1995; de Groot et al., 1998).
U.S. Pat. Nos. 6,162,965 and 6,310,273 disclose that Agrobacterium tumefaciens induces necrosis during co-culture with plant cells, particularly of the Gramineae. Compounds that inhibit necrosis, e.g., 2,5-norbornadiene, norbornene, silver thiosulfate, aminoethoxyvinylglycine, cobalt salts, acetyl salicylic acid, or salicylic acid, may be used to improve transformation efficiency. Similarly, a nucleotide sequence that encodes a cell death suppressor protein (p35, iap or dad-1) may be included in the transformation vector to achieve the same goal.
Similarly, wounding and A. tumefaciens infection of soybean cotyledonary node explants result in extensive enzymatic browning and cell death in the tissue. Thiol compounds, such as L-cysteine, dithiothreitol (DTT), and sodium thiosulfate, improves T-DNA delivery by inhibiting the activity of plant pathogen and wound-response enzymes, such as peroxidases (PODs) and polyphenol oxidases (PPOs). Additive effect was observed with the thiol compounds. The most effective combination for soybean is 1 mM Na-thiolsulfate+8.8 mM L-cysteine+1 mM DTT (Olhoft et al., 2003).
Nitric oxide (NO) has been implicated in defense responses and its control or elimination may increase pathogen infection such as that of Agrobacterium during plant transformation (U.S. Pat. No. 7,388,126). NO modulators include of NG-monomethyl-L-arginine, monoacetate salt (N-Me-L-Arg, AcOH; L-NMMA), NG-monomethyl-L-homoarginine, monoacetate salt (NMMHA, AcOH), NG-monoethyl-L-arginine, monoacetate salt (NMEA, AcOH), NG-monomethyl-L-arginine, di-p-hydroxyazobenzene-p″-sulfonate salt (N-Me-L-Arg, diHABS; L-NMMA), and clinorotation.
Activation of plasma membrane NADPH oxidase is associated with the incompatible interaction of plants with microbes, leading to production of superoxide, which can be converted into H2O2 by superoxide dismutases. NADPH oxidase can be effectively inhibited by diphenylene iodonium (Sagi and Fluhr, 2001). To date, there is no evidence to suggest that NADPH oxidase plays any role in compatible plant-microbe interactions, such as jatropha-agrobacterium interaction during transformation process. Neither exist any report on the use of NADPH oxidase inhibitors, such as diphenylene iodonium, to improve Agrobacterium-mediated plant transformation efficiency.
Overgrowth of Agrobacterium jeopardizes the survival of the transformed plant cells and also has an effect on the T-DNA transfer process. Insertion of multiple copies of a gene of interest into a plant cell is influenced by the frequency of T-DNA transfer into the cell. Agrobacterium-mediated transformation protocols strive to attain transformation events with a limited number of copies of DNA entering into any one cell. The presence of multiple inserts can lead to gene silencing or reduce expression levels of transformed genes, which is caused by several mechanisms including recombination between the multiple copies.
International Publication No. WO 2001/09302 and U.S. Patent Application Publication 2003/0204875 discloses control of Agrobacterium growth during the transformation process in order to improve transformation efficiency. The use of inhibiting agents during inoculation and co-culture of Agrobacterium with a transformable plant cell results, according to the disclosure, in increased transformation efficiencies and a low copy number of an introduced genetic component in several plant systems. Preferred growth-inhibiting agents are compounds containing heavy metals such as silver nitrate or silver thiosulfate, antibiotics such as carbenicillin, and a combination of antibiotics and a clavulanic acid such as augmentin or timentin.
U.S. Pat. No. 6,323,396 claims a process to produce a dicotyledonous transgenic plant by systemically infecting auxotrophic Agrobacterium strain with explant tissue, protoplast or microspore and regeneration on a plant regeneration medium, in the absence of a compound with a bacteriocidal or bacteriostatic effect on the auxotrophic Agrobacterium strain. The Agrobacterium strain is LBA4404metHV (LMG P-18486) or ATHV ade, his, (LMG P-18485). International Publication No. WO/2005/103271 discloses improved method for Agrobacterium mediated transformation of embryogenic suspension culture which is achieved by the use of solid support for co-culturing of the embryogenic cells and a set of media for co-culture and selection of transformants. The method is particularly suitable for transformation of cotton embryogenic calli.